Conventional cancer treatments using radiation and anti-cancer agents inflict substantial burden on human bodies because they widely harm not only cancer cells but also normal cells. The use of conventional cancer treatments using radiation and anti-cancer agents is limited depending on the age and physical conditions of patients. Also, these treatments may not lead to a complete death of remaining cancer cells due to limitations in therapeutic methods. Recently, technology for delivering a drug in a manner specific to cancer cells by using various drug delivery system, such as liposome, bacteria-derived minicells, micelle, polymer-derived nanoparticles, or bacterial outer membrane vehicles (OMVs), has been introduced (Huang S L, Adv. Drug Deliv. Rev. 60:1167-1176(2008), Kim S H, et al., BBA-Biomembranes 1788:2150-2159(2009), Savic R, el al., Science 300:615-618(2003), Torchilin V P., Adv. Drug Deliv. Rev., 64:302-315(2012), MacDiarmid J A et al., Cancer Cell, 11:431-445(2007)). From among these, minicells are bacteria-derived materials and may deliver various drugs (US 2003/0198996 A1), and are receiving much attention due to their high stability and biocompatibility.
Bacterial minicells are a chromosome-depleted cell having a size of about 100 nm to about 400 nm, and do not include chromosome at all. However, RNAs, proteins, cell membrane and wall have the same components or compositions as those of their parent cells. In general, minicell-producing strains can be manufactured by genetically inactivating or over-expressing genes associated with cell division to induce non-equivalent cell division in the course of cell division, and minicell-producing strains are generated from various strains including Escherichia coli, Salmonella typhimurium, Shigella flexnery, Bacillus subtilis, or Listeria monocytogenes (MacDiarmid J A et al., Cancer Cell, 11:431-445(2007)).
When minicells are used for a drug delivery system, lipids and various channel proteins of membrane components can be used in loading or releasing a drug. Also, by changing cell wall or membrane components by using an antibody or an adhesion molecule, cancer-cell targeting and endocytosis into animal cells may be promoted. For example, Escherichia coli derived minicells are coated with a bispecific antibody to increase cancer cell directedness, and when loaded with si/shRNA and an anti-cancer agent and treated sequentially, they show high tumor regression effects with respect to cancer cells that are resistant to anti-cancer agents. (MacDiarmid J A et al., Nat. Biotechnol., 27:643-651(2009)). In this study, however, the minicells derived from S. typhimurium, has lipopolysaccharide (LPS) being an endotoxin as a cell wall component, and when this minicell is used as a drug delivery system in vivo, LPS of the minicell may act as a pyrogen, and unexpected immune responses (inflammation), fever, or the like may occur. To address these problems, from among various human strains (normal flora) that have been evolutionarily adapted, a strain that does not have pathogenicity, easy to culture and genetic modification could be selected in order to use as a drug delivery system.
Corynebacterium glutamicum is one of normal flora strains existing in vivo and is well adapted to the human immune system, and is one of GRAS (Generally recognized as safe) strains and is highly industrially available due to its use in the production of amino acids and nucleic acids (Nakayama K et al., Adv. Exp. Med. Biol., 105:649-661(1978)). Corynebacterium glutamicum is a gram positive strain, does not have an LPS layer in a cell wall, does not form spores, and compared to other wild-type strains, is easily cultured and genetically modified. Accordingly, when Corynebacterium glutamicum is used as a drug delivery system, toxicity problems that may occur from a drug delivery system in itself, for example, LPS of gram-negative bacteria, may be prevented. Moreover, when existing various genetic tools and resources are used, attempt to load drug proteins or target cancer cells may be easily possible.
For genetic modification of Corynebacterium sp., a homologous recombination method using sacB screen system (pK19mobsacB) is used to insert genes into a chromosome or delete them therefrom (Schafer A, et al., Gene, 145:69-73(1994)). Also, shuttle vectors of Escherichia coli and Corynebacterium (pCES208, pXMJ19, or the like) can be used to introduce or express foreign genes. For example, human proteins are over-expressed in Corynebacterium glutamicum (Date M et al., Lett. Appl. Microbiol., 42:66-70(2006)). These disclosures may contribute to the production of various eukaryotic cell-derived drug proteins.
Throughout this specification, many papers and patent documents were used as reference in parenthesis. The cited papers and the disclosures of patent documents are in entirety incorporated into the present specification as reference to clearly explain a level of a technical field the present disclosure belongs to and the content of the present disclosure.